1-adamantyl chalcones for the treatment of proliferative disorders

ABSTRACT

The present invention relates to the compounds of the general formula (I), a composition for and a method of treating breast cancer or other proliferative disorders in a subject using a compound of general formula [I], 
     
       
         
         
             
             
         
       
     
     wherein the substituents are as defined in the specification.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of, and claims priorityto, U.S. patent application Ser. No. 12/506,039, filed 20 Jul. 2009, nowU.S. Pat. No. 8,013,020, which is a divisional application of U.S.patent application Ser. No. 11/400,506, filed 7 Apr. 2006, which is adivisional application of U.S. patent application Ser. No. 10/950,875,filed 27 Sep. 2004, now abandoned, which is a divisional application ofU.S. patent application Ser. No. 10/224,723, filed 20 Aug. 2002, nowU.S. Pat. No. 6,864,264, the entireties of all three of which areincorporated herein by reference as if fully set forth below.

TECHNICAL FIELD

The present invention pertains to novel 1-adamantyl chalcones,compositions containing the novel 1-adamantyl chalcones, and methods fortreating a proliferative disorder using 1-adamantyl chalcones. Moreparticularly, the present invention is directed to a composition for andmethod of treating breast cancer using 1-adamantyl chalcones, alone orin combination with an additional anti-tumor agent.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States. Halfof all men and one-third of all women in the U.S. will develop cancerduring their lifetimes.

Cancer is a condition which develops when abnormal cells in an organbegin to grow uncontrollably, replacing normal tissue. This cellproliferation usually forms tumors, although it may also be blood borne.Cancers may behave differently. For instance, breast cancer may grow ata different rate than lung cancer. Although treatment is usuallydirected to the different characteristics of the particular cancer, mostcancer treatment involves chemotherapeutic agents.

Breast cancer is the second leading cause of cancer deaths among womenin the United States. Second only to lung cancer; breast cancer is theleading cause of cancer deaths among women aged 40 to 55 years. Breastcancer is the most common malignant neoplasm in women worldwide. TheAmerican Cancer Society estimated that in 2001, about 192,200 new casesof invasive breast cancer would be diagnosed among women in the UnitedStates. In addition, an estimated 1500 cases would be diagnosed amongmen. Statistics indicate that in 2001, there were about 40,200 deathsfrom breast cancer in the United States.

The development of breast cancer has long been speculated to be linkedto estrogen stimulation derived from the ovaries. In 1900, Stanley Boyddemonstrated that one-third of the premenopausal women diagnosed withbreast cancer benefited from ovarectomies (removal of ovaries). It isnow known that the reduction of the estrogen levels that accompanyovarectomy is responsible for this effect, and that any form ofendocrine therapy that decreases estrogen levels will benefitapproximately one-third of breast cancer patients with pre- orpost-menopausal breast cancer.

One of the risk factors associated with increased chance of breastcancer development is genetic pre-disposition. A woman with afirst-degree relative with breast cancer is about two to three timesmore likely to develop breast cancer than a woman with a negative familyhistory. Most of the patients with hereditary breast cancer are thoughtto have a mutant BRACA1 or BRACA2 gene. In one study of 33 families withevidence of linkage to BRACA1 or BRACA2 gene, the lifetime risk ofbreast cancer was 87% by age 70.

Breast cancer treatments include surgery, radiation, and chemothereapy.Chemotherapeutic agents and regimens with documented antitumor activityagainst breast cancer, however, have limited success in treating thedisease, as less than one in five patients with stage IV breast cancerare alive five years from the first detection of distant metastases.Although improved response has been observed, overall survival forpatients with metastatic breast cancer has not been significantlyimproved by the progress of the past three decades. Furthermore, despitethe proven benefit of adjuvant systemic therapy in reducing the risk ofrecurrence, a significant fraction of patients with early stage breastcancer still will relapse and ultimately die of metastatic disease.Clearly new active agents and strategies are needed to improve upon thissituation.

It is therefore an object of the present invention to provide novel1-adamantyl compounds for the treatment of breast cancer and otherproliferative disorders.

It is a further object of the present invention to provide novelpharmaceutical compositions of 1-adamantyl compounds for the treatmentof breast cancer and other proliferative disorders.

It is still another object of the present invention to provide a methodfor treating tumors from breast cancer and other proliferative disordersby administering to a subject in need of such treatment atherapeutically effective amount of novel 1-adamantyl compounds.

It is yet another object of the present invention to provide a methodfor treating tumors from breast cancer and other proliferative disordersby administering to a subject in need of such treatment atherapeutically effective amount of novel 1-adamantyl compounds incombination with other anti-tumor agents.

SUMMARY OF THE INVENTION

The present invention relates to the compounds of the general formula(I), a composition for and a method of treating breast cancer or otherproliferative disorders in a subject using a compound of generalformula,

wherein:

R1 is Ad- or Ad-(L1)n-, wherein n is 0 or 1, Ad is adamantyl, and L1 isa linking group selected from the group consisting of C1-6 alkylene,C1-6 cycloalkylene, and C1-6 arylene;

R2 is CY1═CHR3, aryl, aryl optionally substituted by X, or HET;

HET is selected from the group consisting of optionally substitutedpyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, pyrrolyl,pyridinyl, and pyridazinyl, quinolinyl, and thiophenyl, and wherein thesubstitutuent is X;

wherein X is selected from the group consisting of hydrogen, straightchain or branched C1-6 alkyl, halo, amino, C1-6 alkyl amino, C1-6dialkyl amino, pyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, C1-6alkoxy, C1-6 aralkoxyl, aryl, C1-6 aralkyl, nitro, cyano and aphosphorus containing group;

Y and Y1 are independently H, C1-6 alkyl, aryl, or halo, with theproviso that when R2 is CY1═CHR3, then Y is hydrogen;

R3 is selected from the group consisting of aryl, aryl optionallysubstituted by X, or HET;

or a pharmaceutically acceptable salt or derivative thereof.

The compounds of the present invention may also be used alone or incombination with other anti-tumor chemotherapeutic agents for thetreatment of breast cancer or other proliferative disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the Human Estrogen Receptor (ER), which was sequencedfrom MCF-7 human breast cancer cells. The ER protein consists of 595amino acids and has been separated into six different functionaldomains.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used to describe the present invention:

The terms “patient” and “host organism” are used throughout thespecification to describe an animal, preferably a human, to whomtreatment, including prophylactic treatment, with the compounds andpharmaceutical compositions according to the present invention isprovided. For treatment of those infections, conditions or diseasestates which are specific for a specific animal such as a human patient,the terms patient or host refer to that specific animal. In mostapplications of the present invention, the patient is a human.Veterinary applications, in certain indications, however, are clearlycontemplated by the present invention.

The term “therapeutically effective amount” shall mean theadministration of at least one compound according to the presentinvention in an amount or concentration and for period of time includingacute, sub-acute or chronic administration, which is effective withinthe context of its administration for causing an intended effect orphysiological outcome in the treatment of proliferative disorders suchas prostate cancer, lung cancer, pancreatic cancer, breast cancer, coloncancer, ovarian cancer, and bladder cancer. Effective amounts ofcompounds, according to the present invention, include amounts which aretherapeutically effective for delaying the onset of, inhibiting oralleviating the effects of the above disease states. Although effectiveamounts of compounds, according to the present invention, generally fallwithin the dosage range of about 0.1 mg/patient kg to about 100mg/patient kg or more, amounts outside of these ranges, in certaininstances, may be used, depending upon the final use of the composition.

As used herein, the term “alkyl” is defined as any straight-chained orbranched alkyl, including but not limited to methyl, ethyl, propyl,butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, t-butyl,isopentyl, amyl, and t-pentyl.

The terms “proliferative disorder” and “cancer” are used as generalterms to describe any of various types of malignant neoplasms, most ofwhich invade surrounding tissues, may metastasize to several sites, andare likely to recur after attempted removal and to cause death of thepatient unless adequately treated. As used herein, the term cancer issubsumed under the term tumor or neoplasia. Cancers, which may betreated using one or more compounds according to the present invention,include but are not limited to stomach, colon, rectal, liver,pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, prostate,testis, bladder, renal, brain/CNS, head and neck, throat, Hodgkin'sdisease, non-Hodgkin's leukemia, multiple myeloma leukemias, skinmelanoma, acute lymphocytic leukemia, acute myelogenous leukemia, smallcell lung cancer, choriocarcinoma, rhabdomyosarcoma, neuroblastoma,mouth/pharynx, esophagus, larynx, lymphoma and kidney cancer. Compoundsaccording to the present invention, which are used to treat tumorsand/or cancer, are referred to as anti-proliferative.

The term pharmaceutically acceptable salt or derivative is usedthroughout the specification to describe any pharmaceutically acceptablesalt or prodrug form (such as an ester, phosphate ester or salt of anester or a related group) of an adamantyl compound which, uponadministration to a patient, provides directly or indirectly theadamantyl compound or an active metabolite of the adamantyl compound.Pharmaceutically acceptable salt forms of the present compounds are alsocontemplated by the present invention. Nonlimiting examples of suchsalts are (a) acid addition salts formed with inorganic acids (forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid, and the like), and salts formed with organic acidssuch as acetic acid, oxalic acid, tartaric acid, succinic acid, malicacid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginicacid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonicacid, and polygalacturonic acid; (b) base addition salts formed withpolyvalent metal cations such as zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium,and the like, or with an organic cation formed fromN,N-dibenzylethylene-diamine, D-glucosamine, ammonium,tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and(b); e.g., a zinc tannate salt or numerous other acids well known in thepharmaceutical art.

As used herein, the term “HET” represents a mono- or polycyclicheterocyclic or heteroaryl group having ring members selected fromcarbon, nitrogen, oxygen and sulfur. Representative heterocyclic groupsinclude, but are not limited to pyrrolidyl, piperidyl, piperazinyl,morpholinyl, thiomorpholinyl, aziridinyl, tetrahydrofuranyl and thelike. Representative heteroaryl groups include, but are not limited tofuranyl, thiophenyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl,isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyranyl,pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl,1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl,thiadiazinyl, indolyl, isoindolyl, benzofuranyl, benzothiophenyl(thianaphthenyl), indazolyl, benzimidazolyl, benzthiazolyl,benzisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl,quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl,pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl and the like.Heteroaryl is also intended to include the partially hydrogenatedderivatives of the heterocyclic systems enumerated above. Non-limitingexamples of such partially hydrogenated derivatives are2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl,oxazolidinyl, oxazolinyl, oxazepinyl and the like.

The present invention is directed to chalcone derivatives containing anadamantyl moiety and having anti-tumor activity. The compounds aregenerally represented by the formula

wherein R1 is Ad- or Ad-(L1)n-, wherein n is 0 or 1, Ad is adamantyl,and L1 is a linking group selected from the group consisting of C1-6alkylene, C1-6 cycloalkylene, and C1-6 arylene;

R2 is CY1═CHR3, aryl, aryl optionally substituted by X, or HET;

HET is selected from the group consisting of optionally substitutedpyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, pyrrolyl,pyridinyl, and pyridazinyl, quinolinyl, and thiophenyl, and wherein thesubstitutuent is X;

wherein X is selected from the group consisting of hydrogen, straightchain or branched C1-6 alkyl, halo, amino, C1-6 alkyl amino, C1-6dialkyl amino, pyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, C1-6alkoxy, C1-6 aralkoxyl, aryl, C1-6 aralkyl, nitro, cyano and aphosphorus containing group;

Y and Y1 are independently H, C1-6 alkyl, aryl, or halo, with theproviso that when R2 is CY1═CHR3, then Y is hydrogen;

R3 is selected from the group consisting of aryl, aryl optionallysubstituted by X, or HET;

or a pharmaceutically acceptable salt or derivative thereof.

Historical research has focused on the relationship between estrogen andbreast cancer. The research has led to an analysis of the estrogenreceptor (ER).

Nuclear hormone receptors are a family of hormone-activatedtranscription factors that initiate or enhance the transcription ofgenes containing specific hormone response elements. The Human ER, whichbelongs to this family, was cloned and sequenced from MCF-7 human breastcancer cells. The ER protein consists of 595 amino acids with amolecular weight of 66 kDa and has been separated into six differentfunctional domains, as shown in FIG. 1.

Two of these functional domains are highly conserved in the primarysequence of members of the nuclear hormone receptor superfamily (A/B inFIG. 1). One of the domains (C in FIG. 1, the DNA binding domain (DBD),contains two zinc fingers that mediate receptor binding to hormoneresponse elements in the promoters of hormone-responsive genes. In theC-terminal region, the hormone-binding domain (HBD, E in FIG. 1)contains two regions of sequence homology with other hormone receptorsand bestows hormone specificity and selectivity.

The model for estrogen action via the ERα (henceforth referred to as ER)has evolved considerably during the past 40 years. The first realisticconceptual model was proposed by Mueller and colleagues to explain theinitiation of metabolic events in the rat uterus by estrogen. Sincethen, several models have evolved that address the mechanism of how theER functions in the nucleus and how it activates the transcription ofestrogen-responsive genes in the presence of estrogens, an effectdifferentially blocked by antiestrogens.

The six structural domains of the ER are regions that have been definedbased on the putative functions that are contained in each area. The ERcontains two areas called AFs: activation functions-1 (AF-1) is locatedin the amino-terminal region of the ER, and activation functions-2(AF-2) is located in the carboxyl-terminal region in the ligand bindingdomain (LBD) of the ER; these are synergistic when the ER is activatedby estrogen. AF-1 and AF-2 are autonomous in that they are located atthe N- and C-termini, respectively. Activation function-1 (AF-1) isthought to be responsible for the promoter-specific transcriptionalactivation independent of the presence of ligand and AF-2 providesligand-specific activation.

The C region contains the DBD and a dimerization domain. The DBD is themost highly conserved region in the nuclear hormone receptorsuperfamily. The DBD consists of two zinc fingers that fold into twohelical domains upon the coordination of one zinc to four cysteines anda third helix that extends from the zinc fingers. These zinc fingers areessential components of the ER because when the ER lacks the DBD, itcannot bind DNA in vitro or in vivo. However, the C region alone is notsufficient to bind an Estrogen Response Element (ERE). The A/B regioncan be deleted without compromising the DNA binding ability but deletionof the basic amino acids (amino acids 256 to 270) located down-stream ofthe zinc fingers does impair the ability of the receptor to bind EREs.

There are many similarities in the zinc finger regions among differentsteroid hormone receptors, but there are precise differences thataccount for the specificity of each receptor. It is believed that thespecificity of a certain receptor is afforded by the first of the twozinc fingers. These conclusions are based on mutagenesis (changes of Cysto Gly) in the region of the first finger. The results prove that thereceptor binds to specific nucleic acid residues in the major groove ofthe DNA helix. The second zinc finger is responsible for stabilizingthis interaction through ionic bonds with the phosphate groups in theDNA backbone. In addition to these mutational studies, domain-swappingexperiments in which the ER DBD was exchanged with the DBD of theglucocorticoid receptor showed that the chimeric protein activatesglucocorticoid responsive genes in the presence of estrogen.

Estrogen diffuses through the plasma membrane of cells where it binds tothe ER. For many years, it generally was thought that estrogen bound tothe ER in the cytoplasm and translocated into the nucleus, but it isknown now that the ER is a nuclear transcription factor that initiallyinteracts with estrogen in the nucleus. Once estrogen binds to the ER, achange in conformation and homodimerization occurs.

Although phosphorylation of steroid hormone receptors enables them tobecome transcriptionally active, until recently, the role ofphosphorylation of the ER was still in question. Phosphorylation of theER from MCF-7 and calf uterus is estrogen-dependent and, in addition,increases the receptor's affinity for specific DNA sequences. The basallevel of ER phosphorylation increases three-to four-fold upon treatmentwith estrogen and antiestrogens. However, the key to elucidating themechanism of estrogen action is the identification of the selectivesites for phosphorylation. Several serines in the amino-terminal portionof the human ER may play a role in hormone-regulated phosphorylation.However, when phosphopeptide maps of wild-type and mutant ERs treatedwith estrogen or antiestrogens are compared, the results are similarindicating that differential phosphorylation between these receptorscannot account for any differences in function. An alternate approachmight be the identification of enzymes responsible for phosphorylation.There are several protein kinases thought to be involved inphosphorylation of the ER (ER kinase, protein kinase C. protein kinasesA, and casein kinase II). Recently, a mitogen-activated protein kinasealso was implicated in phosphorylation of the ER on Ser 118 resulting inthe activation of ER AF-1. Interestingly, another consequence ofphosphorylation of the ER is the regulation of homodimerization throughphosphorylation of tyrosine 537.

Although phosphorylation may play a part in receptor activation,exciting progress has been made in understanding how the receptorcooperates with other proteins to assemble a transcription unit for geneactivation. The receptor can be viewed as a skeleton to assemble theunit as a prelude to DNA unwinding and the transcription of selectedmRNAs. To achieve this, the receptor eventually must interact with otherproteins as well as bind to one or several EREs.

As stated above, the ER contains two areas called AFs: AF-1 is locatedin the amino-terminal region of the ER, and AF-2 is located in thecarboxyl-terminal region in the ligand binding domain (LBD) of the ER;these are synergistic when the ER is activated by estrogen.Katzenellenbogen and colleagues used mammalian cells to show that theAF-1 and AF-2 regions, when expressed as separate polypeptides,functionally interact in response to estrogen and antiestrogens. Theyfound that this interaction could activate transcription in response toestrogen. In addition, when mutations were made in AF-1 or AF-2, thefunctional activity of these domains was inhibited, and notranscriptional activity was seen. Additionally, when mutations weremade in the LBD, estrogen binding was eliminated and no transcriptionalactivity could be detected. These experiments suggested that estrogenbinding to the ER facilitates a conformational change that brings AF-1and AF-2 in direct association with one another leading to synergy thatresults in transcriptional activation. These experiments provide amechanistic explanation for the role of the two AFs in mediatinghormone-regulated transcription.

In addition to understanding the mechanism through which the ER becomestranscriptionally active, many of the amino acids important in thebinding of ligand to the ER have been identified. Harlow and coworkersshowed a covalent attachment between Cys530 and both estrogen agonistand an antagonist. This work also suggested that other cysteine residuespresent in the LBD might be important for ligand-mediatedtranscriptional activation. Further mutant ERs have been constructedwith mutations at the other cysteine residues present in the LBD. Eachof these mutants showed an affinity similar to that of the wild-type ER.When these mutants were tested in reporter assays, the mutants C530A andC530S showed unaltered binding to estrogens and antiestrogens, but thetransactivation response to both estrogens and antiestrogens hadchanged. After showing that the C530 is involved in discriminatingbetween ligands, Pakdel and Katzenellenbogen examined the role of aminoacids adjacent to the other cysteines in the LBD of the ER. The resultsshowed that the amino-terminal domain of the LBD was important indifferential transcriptional activation but not in binding affinity.When the carboxyl-terminal region of the LBD is mutated, this rendersthe protein transcriptionally inactive although it can still bindligand, making this a very powerful dominant negative ER. Thus, there isa distinction between the hormone binding and the transactivationfunctions.

Once the ER has bound estrogen and dimerized, it binds to EREs presentin the promoter region of genes. These EREs are 13 base pair palindromicsequences located upstream from the transcriptional start site. The EREsfunction by enhancing the transcriptional potential of gene. EREs havebeen identified and defined using reporter systems to test the enhancerability when exposed to different compounds. Also, deletional analysishas allowed the definition of the sequence of EREs. Optimally, itconsists of two inverted repeats separated by any three base pairs. Theexact sequence of EREs varies between species and genes.

Some models of estrogen action predict that when the dimerizedhormone-receptor complex binds to the palindromic ERE, it forms a loopedstructure allowing the ER to interact with the transcriptional apparatusat the RNA initiation site. It is thought that the hormone-receptorcomplex can recruit components of the transcriptional complex and servesas a nucleation site. Previous studies focus on the interaction of theER with EREs, but more recently, there has been a shift toward the studyof ER receptor interactions with ancillary proteins in the nucleus.

Several chemotherapeutic anti-tumor agents are believed to function bythe interaction of the ER with EREs or ancillary proteins in thenucleus.

Tamoxifen, a nonsteroidal antiestrogen, is the most commonly usedchemotherapeutic agent for treatment of advanced breast cancer and asadjuvant therapy in premenopausal women. Currently, tamoxifen isubiquitously used in hormone therapy against post menopausal breastcancer. Clinical trials for long term therapy with tamoxifen as apreventive measure against breast cancer have demonstrated itseffectivness. Tamoxifen is effective against metastatic breast cancer,delays relapse and has been shown to prolong survival after primarysurgery.

Antiestrogens can be classified into two major groups: analogs oftamoxifen or its metabolites, including 4-Hydroxytamoxifen (4-OHT),which have mixed strogenic/antiestrogenic actions in laboratory assays.

The triphenylethlyene structure of tamoxifen has provided the basis forseveral new analogs that are being investigated in clinics. The findingthat tamoxifen is metabolized to 4-Hydroxytamoxifen, a potentantiestrogen, also has provided a central theme for drug development.

Toremifene, or chlorotamoxifen, has been investigated thoroughly as anantiestrogen and antitumor agent in the laboratory, and currently isbeing used for the treatment of advanced breast cancer and is beingtested as an adjuvant therapy. Toremifene is of interest because it doesnot produce DNA adducts in rat liver and, as a result, is not a potentcarcinogen in rat liver.

Idoxifene is a 4-iodopyrrolidino derivative of tamoxifen that hasantiestrogenic and antitumor properties in laboratory rats. Idoxifene isa metabolically stable analog of tamoxifen synthesized to avoid toxicityreported with tamoxifen in the rat liver. Substitution of halogens inthe 4-position of tamoxifen is known to reduce antiestrogen potency bypreventing conversion to 4-OHT, and it was argued that reduceddemethylation of the side chain also would avoid the formation offormaldehyde in the liver.

Droloxifene, or 3-hydroxytamoxifen, has been studied extensively as anantiestrogen and an antitumor agent in the laboratory. This drug doesnot form DNA adducts under laboratory conditions, or produce livertumors in rats. Extensive clinical testing has shown activity in thetreatment of advanced breast cancer in postmenopausal patients.

TAT-59 is a prodrug that is being developed for the treatment ofadvanced breast cancer. TAT-59 has been shown to inhibit the growth ofER-positive, DMBA-induced rat mammary carcinomas. The drug inhibits thegrowth of estrogen-stimulated, ER-positive breast cancer cellstransplanted into athymic mice. The drug is activated metabolically to adephosphorylated form that binds with high affinity to the ER. Clinicalstudies using TAT-59 for the treatment of advanced breast cancer havenot been published.

Additionally, compounds are being investigated that do not resembletriphenylethylenes but do exploit the known structural requirements forhigh binding affinity for the ER.

The compounds LY117018 and raloxifene have high binding affinity for theER but a lower estrogenic activity than tamoxifen when using rodentuterine assays. They are competitive antagonists of estrogen action butalso can block the estrogen-like effects of tamoxifen in the uterus.This demonstrates a single mechanism of action for this class of drugsthrough the ER.

Although tamoxifen has been used successfully to treat approximately 70%of all ER-positive tumors, there are some disadvantages involved withits use: 1) an increased incidence of endometrial cancer; 2) itsmechanism of action almost exclusively lends itself to treatment ofER-positive breast tumors; and 3) with constant treatment of tamoxifen,resistance can develop. Side effects from tamoxifen treatment have beenobserved in clinical trials. Although it increases hot flashes, it maybe beneficial in preventing coronary heart disease because it lowerslow-density lipoprotein cholesterol and apolipoprotein B. Not everythingunder estrogen control is affected by tamoxifen. For example, bone massand clotting factors remain unchanged. On the other hand, long termtreatment with tamoxifen can induce growth of hormone responsive humanbreast tumor in nude mice. In nude mice with breast tumor andendometrium tumor xenografts, treatment with tamoxifen results in adecrease in size of the breast tumor, but an increase in size of theendometrial tissue. Long term exposure to tamoxifen has no effect on thedoubling time of the estrogen receptor negative cell line MDA-MB435, butit induces the formation of a new tetraploid clone that, when implantedin the flank of nude mice yields a greater tumor mass that has a highermitotic index and is more anaplastic than tumors obtained from wild typeMDA-MB435 cells.

Genistein, a natural flavanone compound found in soy, has been proposedto be responsible for the low rate of breast cancer in Asian women.Genistein is a tyrosine kinase inhibitor that works by altering theauto-phosphorylation step on the Epidermal Growth Factor Receptor(EGFR). Genistein is found to exert pronounced antiproliferative effectson both ER-positive and ER-negative human breast carcinoma cells throughG2-M arrest, induction of p21Waf1/CIP1 expression, and apotosis.

Genistein also suppresses the production of stress proteins in cells;such as heat shock proteins and glucose-regulated proteins that normallyhelp cancer cells survive destruction by the immune system.

Doxyrubicin is a common chemotherapeutic agent used for treatment ofvarious types of cancers including breast cancer. It is categorized asan anthracycline antibiotic agent that exerts its effect on cancerousand normal cells by binding to the DNA through intercalation betweenspecific bases and blocking the synthesis of new RNA or DNA (or both).This intercalation causes DNA strands scission, and interferes with cellreplication.

One of the major problems associated with doxyrubicin treatment istoxicity symptoms such as myelosuppresion, and cardiac toxicity.

Paclitaxel (Taxol®), the prototype taxane, was developed as ananticancer agent at an accelerated pace after the elucidation of itsunique mechanism of action and by the initial demonstration of itsactivity in refractory advanced ovarian carcinoma. The drug functions asa mitotic spindle poison through the enhancement of tubulinpolymerization. Multidisciplinary efforts to procure sufficientquantities of this agent have allowed a rapid expansion of clinicaltrials.

Taxotere is an anticancer agent that inhibits cancer cell division byessentially “freezing” the cells internal skeleton, which is made up ofelements called microtubules. These microtubules assemble anddisassemble during the cell cycle, but Taxotere promotes the assemblyand blocks the disassembly, thus preventing cancer cells from dividing.This action can lead to cancer cell death. The observed side-effect is areduction of the white blood cell count.

Aredia is used for treatment of patients with osteolytic bone metastasesof breast cancer, in conjunction with standard antineoplastic therapy.It exhibits its clinical usefulness by inhibiting bone resorbtion. It isparticularly useful for the treatment of hypercalcemia associated withmalignancy. It has been proven to reduce the incidence of skeletalcomplication of metastatic breast cancer. However, the incidence andtype of adverse events with Aredia were fatigue, fever, nausea, skeletalpain, transient arthralgias, and myalgias.

Arimidex is used for advanced breast cancer in postmenopausal womenwhose disease has progressed following therapy with tamoxifen. Many sideeffects have been reported including asthemia, nausea, hot flashes,pain, and back pain.

Navilbine is a chemotheraputic drug available for the treatment ofmetastic breast cancer. It is indicated for the treatment of patientswith metastatic breast cancer who have failed standard first-linechemotherapy. Side effects include tingling in fingers and toes,constipation, and hair loss.

Chalcone 16 (1,3-Diphenyl-2-propen-1-one) contains aromatic rings (A andC) linked by an olefinic bond and a keto group (B).

Derivatives of Chalcone 16 are widely distributed in higher plants, andplay a central role in the biosynthesis of flavonoids. Chalcone 16itself is considered to be a flavonoid, although its chemical structureis different from that of other flavonoids, which have the 2-phenylchromone structure 17.

Chalcone 16 has a stilbene configuration in which two phenyl groupsbracket an α,β-unsaturated carbonyl group. Amongst stilbene derivativesare a variety of synthetic compounds, for example tamoxifen, awidely-used agent for the treatment of breast cancer, which bind tosteroid hormone receptors. Compounds containing the α,β-unsaturatedmoiety (a reactive chemical species) have been found to bind toreceptors that increase the activity of phase II enzymes that metabolizexenobiotic.

The similarity between chalcone and tamoxifen is that both exhibit anene functionality that separates the phenyl groups. It is important forthe structure to have this functionality, which is the site for thebinding.

The vast majority of derivatives of chalcone 16 have been shown toinhibit lipoxygenase activity and TPA-induced tumor promotion of themouse epidermis. Isoliquiritigenin 18 (4,2′,4′-trihydroxychalcone) isparticularly potent in this regard. The biological effects produced bychalcone 16 containing other types of substitutions have also beeninvestigated.

In one such study,(E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-1-propenyl]benzoic acid 19 wasfound to induce differentiation of HL-60 leukemia cells andtetratocarcinoma cells.

In another study,(2E)-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2-propen-1-one20 and related compounds were reported to induce antimitotic activityagainst tumor cells in vitro.

Lichochalcone A 21 has been shown to inhibit tumor promotion in mice aswell as exhibit antitumor activity against L1210 leukemia and B16melanoma cells in vivo.

More recently, 3′-methyl-3-hydroxychalcone 22 has been reported to be apotent inhibitor of proliferation of several lines of malignant humancells in vitro, and to suppress TPA-induced tumor promoting activity inmouse skin in vivo.

An additional attribute of this chalcone 22 is its capacity to inhibitthe binding of estradiol to type II estrogen binding sites in HGC-27cells. Thus, several substituted chalcones have been shown to haveeffects such as inhibition of cell proliferation and tumor promotionthat might endow them with chemopreventive properties.

Retenoids 23, N-phenylbenzamide, which are structurally similar tochalcone, have been shown to exhibit inhibitory activity againstnon-small cell lung carcinoma (NSCLC).

Additionally, certain quinoxalines 24 and quinolines 25 show promise asinhibitors of tumor growth.

Certain derivatives of chalcone 16 and some flavones have shown promiseas antitumor agents in breast and other cancers. For example, apigenin26 and some of its congeners were reported to possess antiproliferativeactivity against the human breast cancer cell line ZR-75-1.

6,4′-Dihydroxyflavone 27 has a binding affinity for the ER, and flavonederivative 28 (BE-14348B) exhibits strong estrogenic activity.

The ER binding affinity and the estrogenic activity were also reportedfor the isoflavone derivative daizein 29.

Recently compound (L86-8275) 30 was reported to exhibit antitumoractivities against several types of human breast cancer cell lines.

Chalcone 16 is widely consumed by herbivorous animals, and it is nottoxic. Derivatives of chalcone 16 seem to have pharmacologicalproperties such as estrogenic activity, inhibition of inflammation andanti-microbe activity, and it has also been reported to have aninhibitory effect on tumor growth.

Many derivatives of adamantane are biologically active compounds with abroad spectrum of activity. The spatial structure, hydrophobicity, andlipophilicity of adamantane ensure favorable conditions for itstransport through biological membranes. The introduction of an adamantylmoiety into organic compounds changes and often enhances theirbiological activities.

A review of the chemical literature revealed that only two compoundscontaining the adamantane moiety have been reported to exhibit activityagainst breast cancer. These two compounds are: the acid6-[3-(1-adamanty0-4-hydroxyphenyl]-2-2-Naphthalenecarboxylic Acid(CD437, AHPN) 39, and the corresponding disubstituted admantyl ester,6-[3-(3,5-dimethyl-1-adamantyl)-4-methoxyphenyl]-2-naphthoate 40.

These compounds 39 and 40 have also shown activity against other formsof cancer, especially leukemia.

To our knowledge no study concerning the anti-breast cancer activity ofchalcone and chalcone-like derivitives containing the adamantyl moietyhas been reported in the current available literature.

The synthetic scheme for the series of adamantyl chalcone andchalcone-like compounds was to prepare derivatives with structuralmodifications in three regions of the basic chalcone 16 skeleton, (A, B,and C). By altering these basic regions, our laboratory was able toexamine some of the substituent effect modifications that modulatebiological activity and change potencies of these compounds towardshuman breast cancer cell lines MCF-7 and MDA-MB435.

Synthesis of the Preferred Embodiments

Preferred compounds of the present invention were conveniently dividedinto four categories: 1) Series I. Chalcone-like compounds with thearomatic A ring replaced by the adamantane moiety; 2) Series II.Chalcone-like compounds with modification in the A ring and B regions;3) Series III. Chalcone-like compounds with the aromatic C ring replacedby heterocycle group; and 4) Series IV. Chalcones with the adamantanemoiety substituted on the aromatic A ring. The synthesis andcharacterization of each series will be discussed in the section thatfollows.

Series I. Synthesis of Chalcone-Like Compounds 43 with Aromatic A RingReplaced by Adamantane Moiety

A series of twelve chalcone-like compounds 43 with aromatic A ringreplaced by the adamantane moiety was prepared by base-catalyzed Aldolcondensation of 1-adamantyl methyl ketone 41 and substitutedbenzaldehydes 42 (eq 1).

1-Adamantyl methyl ketone 41 reacted readily, at ambient temperature,with a variety of substituted benzaldehydes 42 in alkaline ethanol toafford the corresponding chalcone like compounds 43(a-1) in good yields.Compounds synthesized by this method are shown in Table 1. Thesecompounds were synthesized, purified and fully characterized by NMR,GC-MS, and Elemental Analyses.

Table 1 shows that the percent yield of products tends to be very goodto excellent regardless of the substituent on the aromatic ring. Theease with which the compounds were obtained makes this particularreaction a very convenient method for its application in the synthesisof widely varying analogs with potential antineoplastic activity andother biological activities.

Literature reviews and observations in our laboratory suggest that themelting points of adamantane derivatives tend to vary by several degreesaccording to method of determination (heating rate, sealed capillary, orcover glass). To ensure consistency melting points were determined on acalibrated programmable Electrothermal 9200 Melting Apparatus with setpoint usually 5° C. below predetermined (estimated) melting point and aramp rate of 0.3° C./min This method enabled the accurate determinationof melting points within one-tenth of a degree.

TABLE 1 Chalcone-Like Compounds 43 Synthesized with Aromatic A RingReplaced by the Adamantane Moiety Compd X % YIELD MP ° C. 43a H 9290.1-91.4 43b p-CN 96 160.1-161.3 43c p-NO₂ 68 165.0-166.1 43d p-Cl 89141.8-143.3 43e p-N(CH₃)₂ 92 155.1-156.2 43f p-CH(CH₃)₂ 94 93.5-94.9 43gp-OCH₃ 96 114.3-115.0 43h p-F 94 129.8-131.4 43I o-Br 91 120.3-121.6 43jp-OCH₂Ph 90 132.7-134.7 43k p-Ph 86 133.6-135.0 43l p-Et 88 78.3-79.9

Proton NMR data for H_(a) and H_(b) (structure 44) were examined todetermine trends in chemical shifts as well as to determine thestereochemistry of the double bond. The proton NMR chemical shifts (δ,ppm) and coupling constants (J_(ab), Hz) for H_(a) and H_(b) are givenin Table 2.

Table 2 shows that the chemical shift for H_(b) varies from 6.98 to 8.08ppm depending upon substituent X on the aromatic ring. Electronwithdrawing groups tend to give signals at lower field (for instanceNO₂, 7.28; F, 7.64 ppm) whereas electron donating groups show signals athigher field (N(CH₃)₂ 6.98, CH(CH₃)₂ 7.15 ppm).

The values for the coupling constants J_(ab) for protons H_(a) and H_(b)are consistent with trans stereochemistry. It is widely known that forthree-bond proton-proton coupling, the coupling constant J_(ab) dependson the dihedral angle φ between H_(a) and H_(b). This dependence of thecoupling constant J_(ab) on dihedral angle φ is described by the wellknown Karplus-Conroy curve. For cis protons H_(a) and H_(b) on a doublebond in alkenes, the dihedral angle φ is zero degrees (0°). Similarly,for trans protons Ha and H_(b) on a double bonds in alkenes, thedihedral angle φ is one hundred and eighty degrees (180°). A review ofthe Karplus-Conroy curve revealed that it predicts larger couplingconstants for trans II_(a) and II_(b) stereochemistry about a doublebond in alkenes. Consistent with the Karplus-Conroy curve prediction,experimental measurements have shown that the coupling constant J_(ab)for cis protons H_(a) and H_(b) usually fall within the range of 9-12Hz, while the coupling constant J_(ab) for trans protons H_(a) and H_(b)generally fall within the 14-17 Hz range. The proton coupling constantsJ_(ab) for H_(a) and H_(b) in compounds 43(a-1) vary from 15.4 to 15.9Hz, all within the range for a trans substituted double bond. Therefore,we have assigned a trans configuration for these compounds.

TABLE 2 Proton NMR Chemical Shifts and Coupling Constants for H_(a) andH_(b) (43) Chemical Shift Coupling (ppm) Constant J_(a-) Compd X H_(a)H_(b) _(b)(HZ) 43a H 7.69 7.18 15.6 43b p-CN 7.67 7.23 15.6 43c p-NO₂7.68 7.28 15.7 43d p-Cl 7.62 7.14 15.6 43e p-N(CH₃)₂ 7.67 6.98 15.4 43fp-CH(CH₃)₂ 7.69 7.15 15.9 43g p-OCH₃ 7.65 7.05 15.4 43h p-F 7.64 — 15.643I o-Br 8.08 7.12 15.7 43j p-OCH₂Ph 7.67 7.07 15.5 43k p-Ph 7.74 7.2315.4 43l p-Et 7.67 7.18 15.5

Two compounds in this series 43f and 43g were successfullyrecrystallized for crystal structure determination. The crystallinestructures obtained for these two compounds supported the stereochemicalassignment made by the NMR. The conformation of the styrene subunit withrespect to the carbonyl for 43f is trans with 01-c1-c2-c3 andc1-c2-c3-c4 torsion angle values of −11.8 and 176.0, respectively.Likewise the conformation of the styrene subunit with respect to thecarbonyl for 43g is trans with 01-cl-c2-c3 and c1-c2-c3-c4 torsion anglevalues of −4.7 and 179.4, respectively.

The fragmentation pattern is very consistent for all these compounds.The base peak observed at m/e 135 (except for the N,N-dimethyl andmethoxy derivatives) arises from the decomposition of the adamantylcation. This fragmentation of adamantane is very common in 1-substitutedadamantanes. However, the parent peak, if it appears at all, is veryweak.

Series II. Chalcone-Like Compounds 45 with Modification in A and BRegions

A series of chalcone-like compounds 45 with modification in the A and Bregions was prepared by based-catalyzed Aldol condensation of1-adamantyl methyl ketone 41, with substituted cinnamaldehydes 44 inalkaline ethanol at ambient temperature to give the correspondingchalcone-like compounds 45 (eq. 3). The percent yield and melting pointsfor these compounds 45 are presented in Table 3.

TABLE 3 Chalcone-Like Compounds 45 Synthesized with Modification in theA and B Regions Compd X Y % YIELD MP ° C. 45a OCH₃ H 71 188.0-190.0 45bH CH₃ 74 158.8-160.3 45c H CL 68 171.2-172.1

Series III. Chalcone-Like Compounds 47 with Aromatic C Ring Replaced byHeterocycle Group

A series of chalcone-like compounds 47 with modification in the A and Cring was synthesized. Het is used to represent the heterocyclic group.The reaction was carried out via aldol condensation of 1-adamantylmethyl ketone 41 and the heterocyclic aldehyde 46 to afford the desireproduct 47 as shown in (eq 4). The percent yield and melting point forthese compounds 47 are presented in Table 4.

TABLE 4 Chalcone-Like Compounds 47 Synthesized with Aromatic C RingSubstituted by Heterocyclic Ring Compd HET % YIELD MP ° C. 47aPyrid-2-yl 72 94.4-97.5 47b Pyrid-3-yl 81 105.7-106.9 47c Pyrid-4-yl 85125.7-127.5 47d 6-methylpyrid-2-yl 54 119.1-120.3 47e Quinol-4-yl 64120.8-122.0 47f Quinol-3-yl 71 157.0-159.0 47g Quinol-2-yl 39165.0-167.0 47h Thiophen-2-yl 68 197.2-98.3 Series IV. Synthesis of Chalcones 58 with Adamantane Moiety Substitutedon Ring A

This series of compounds was prepared to study the effect of theadamantane moiety as a substituent on the aromatic A ring. In order todo this we had to prepare the appropriate aryl adamantanes prior tocarrying out the Aldol condensation.

Synthesis of Aryl Adamantanes 50 Adamantylation of aromatics has notbeen frequently studied, in spite of its potential importance. Testafariand coworkers studied adamantylation of aromatics in a radical reactionwith the 1-adamantyl radical to describe the effect of substituents onthe reactivity and isomer distribution. Newman first reported theAlCl₃-catalyzed Friedel-Crafts alkylation of benzene with1-bromoadamantane, where a complex product mixture was obtained (eq 5).

Later the adamantylation of reactive aromatics with adamantyl nitrateand a FeCl₃-catalyzed reaction was described using an excess of thearomatic compound. In the case of toluene as substrate exclusiveformation of para-adamantylated product was reported. The study ofFriedel-Crafts adamantylation of benzene and toluene in the presence ofboron tris(triflate) has also been reported (eq 6).

All attempts at adamantylating nitrobenzene were unsuccessful, asexpected. The nitro substituent is a strong electron withdrawing groupthus greatly decreasing the reactivity of the aromatic system towardselectrophiles.

Iron filing-catalyzed Friedel-Crafts alkylation of aromatic compoundswith 1-bromoadamantane 48 is unknown. However, the use of iron as aFriedel-Crafts catalyst in alkylation of aromatic systems has beendescribed in the literature. In this study, a novel methodology for theadamantylation of benzene 49 and some of its derivatives has beendiscovered. One of the most remarkable features of this reaction is theease of product separation from the iron catalyst by simple filtration.Moreover, no usual aqueous basic work-up is necessary.

This particular adamantylation reaction appears to be versatile as towhat type of catalyst can be used. Iron (0), Iron (II) oxide and iron(III) oxide does give the Friedel-Crafts products in very good yields.

TABLE 5 Some 1-Aryladamantanes 54 Synthesized By this Method Compd X %YIELD Mp ° C. 54a H 90 87.3-88.9 54b Me 93 95.3-98.9 54c OMe 9578.4-81.3

Given that these Friedel-Crafts type alkylation reactions are expectedto proceed via cationic intermediates, then it is reasonable to expectFe to be an effective catalyst. 1-aryladamantanes 54 were prepared byheating (boiling temperature of the reacting aromatic system) thereaction of 1-bromoadamantane 48 with substituted benzenes 53 in thepresence of Fe (eq 7). The experimental results show that the reactionrates decrease significantly in parallel with the decrease in reactiontemperature. The adamantylation reaction does not take place at roomtemperature.

As evidenced by the experimental data presented in Table 5 thissynthesis indeed produced very pure product in excellent yields. Themechanism for this reaction appears to be ionic as confirmed by thestereochemistry of the Friedel-Crafts alkylation products. For instance,electron withdrawing substituents on the aromatic system yieldexclusively the meta-substituted derivatives, whereas electron donatingsusbtituents afford the corresponding para-substituted products. It isworth noticing that only the para substituted isomer is observed. Thesignificant differences in the stability of ortho and para isomers canbe explained by the strong steric interaction between the orthosubstituent and the bulky adamantyl group. Thus preventing the formationof the ortho isomer. These products were fully characterized by GC-MS,proton and carbon-NMR. The results described herein also agree withmonoadamatylated products synthesized by Olah et al. In addition, Engelet al found the formation of bi-adamantane in yields of at least 20%when forcing the mechanism to follow the free radical pathway in thepresence of AIBN and tributyltin. These reagents are well known toinduce the formation of free radicals. In this work, no bi-adamantanewas detected thus indicating that mechanism for the Fe catalyzedFriedel-Crafts adamantylation proceeds via ionic pathway.

It is well known that 1-adamantyl cation is a stable bridge-headcarbocation. This carbocation is formed by reacting bromoadamantane witha Lewis acid catalyst and attacking by the aromatic system in atraditional Friedel-Crafts way. The substitution pattern will determinewhether the mechanism is via a free radical or ionic. A series of simpleexperiments were designed in order to study the stereochemistry of theproducts thus determining the preferred mechanistic pathway.

Electron donating substituents favor meta substitution on the aromaticring when the reaction is proceeding by a free radical mechanism. Inaddition, electron withdrawing groups favor ortho/para substitution whenfollowing similar pathway. However, electron donating substituents favorortho/para substitution and electron withdrawing substituents favor metasubstitution when the reaction takes place via an ionic mechanism.

Solid acid catalyzed Friedel-Craft adamantylation reactions have beenfound to be a convenient methodology for the preparation ofadamantyl-substituted aromatics with high para-regioselectivity.DeMeijere et al. has utilized palladium/charcoal mixed with potassiumcarbonate as catalyst for Friedel-Crafts adamatylation reactions. Histeam of researchers has proved the mechanism for such reaction is ionic.However, the times for the reactions to take place tend to be mildlylengthy (from 12 to 24 h) and in some instances, regardless of theelectronic nature of the substituent on the aromatic system, some of thedesired products are not formed.

The methodology described in this work makes use of much milderconditions affording high yields of adamantylated products. The ironcatalyzed Friedel-Crafts adamantylation presented herein does have agreat advantage over the literature procedure due to the facility ofhandling the reactants, the relatively low prices of the startingmaterials, and the easyness in extracting the final products with highdegree of purity (in some instances, crystals form after filtration andwashings with hexane). An example is compound 54c.

The fragmentation pattern for 1-arylsubstituted adamantanes follows adifferent process than that of 1-alkylsubstituted adamantanes. Usually,and in these cases all three compounds follow the same behavior,adamantane fragments itself into an aromatic (benzene) unit losing aC₄H₉ fragment (m/e 57), thus leaving behind a benzene-aromatic fragmentas a base peak.

Synthesis of 5-(1-adamantyl)-2-methoxy Acetophenone 55. Thisacetophenone 55 was synthesized from the anisole 54c by Friedel-Craftsacylation prior to preparation of the chalcone derivatives by Aldolcondensation. Friedel Crafts acylation is known to perform well if thesubstituent is an electron donating group. Compound 54c was selectedsince it best exhibited this quality.

The 1-aryladamantane 54c was converted to5-(1-adamantyl)-2-methoxy-acetophenone 56 by treatment with acetylchloride in the presence of AlCl₃ as a catalyst at room temperature (eq.9)

Synthesis of the Chalcone 58. After considering that the resonanceeffect in aromatic compounds increase biological activity, chalcone 58was synthesized. Treatment of the resulting product 55 with biphenylbenzaldehyde 57 as described previously to give 58 (eq 10).

In this study we have synthesized a wide variety of compounds and haveidentified several chalcone-like compounds, which possessgrowth-inhibitory activity against selected cell lines, particularlybreast cancer cell lines. Some of the chalcone-like compounds (seriesIII), which have quite different chemical structures from conventionalchalcones, show higher activity against breast cancer cell lines thanchalcones. Screening of these compounds on breast cancer cell lines hasbeen conducted in vitro, and the data are presented in the sections thatfollow.

Two types of antiestrogens are the analogs of tamoxifen or structuralderivatives of the triphenylethylene type of drug. All of thesecompounds are inhibitors of the binding of E2 to the ER, but there thesimilarity ends. Antiestrogens seem to form a receptor complex that isconverted incompletely to the fully activated form. As a result of theimperfect changes in the tertiary structure of the protein, the complexis only partially active in initiating the programmed series of eventsnecessary to orchestrate gene activation.

Studies in vitro demonstrate that very low concentrations oftriphenylethylene-type antiestrogens can cause a single round ofreplication in breast cancer cells, but high concentrations of theseantiestrogens are completely inhibitory. It is possible that the modestpartial estrogen-like action at low concentrations causes the tamoxifenflare that sometimes is observed when therapy is started in patientswith bony metastases. Once steady-state levels of the drug have beenachieved (approximately 4 to 8 weeks with 20 mg/day), symptoms will havedisappeared and the patient will experience a response to therapy. It isimportant therefore, to be able to identify tumor flare and notprematurely terminate a beneficial therapy. Nevertheless, a recentreport has demonstrated that clinicians often prematurely terminateantiestrogen treatment based on changes in bone scintigraphymisinterpreted as progressive disease. Because there are toxicologicaladvantages in disease control with antiestrogens, a premature change tochemotherapy may be inappropriate.

The interactions of the ER with EREs also depend on the nature of theligand to which it has bound. When the effects of binding of estrogenicand different antiestrogenic ligands to an ERE are quantitated, it wasfound that E2-ER and 4-OHT-ER complexes bound a singlet ERE with similaraffinity. However, at saturation, 4-OHT-ER binds 50% the level of E2-ERbinding. When the tandem copies of EREs were tested, E2-ER exhibitedcooperative binding whereas 4-OHT-ER displayed little or nocooperativity. Therefore, specific ligand binding can alter bindingaffinity of the ER to DNA and the amount of receptor that is saturatedpresumably by inducing different conformations in the ER protein.Further studies of the mechanism through which antiestrogens antagonizethe transcription of estrogen-responsive genes through differentialbinding to EREs show that the flanking sequences and stereoalignment ofEREs are important.

A further investigation of antiestrogenic ligands demonstrated that when4-OHT-ER binds to DNA one molecule of 4-OHT dissociates from the ERdimer. Under the same conditions, tamoxifen aziridine, which covalentlyattached to the ER, show a binding stoichiometry identical with that ofE2-ER, which is one dimeric receptor per ERE compared with one monomerof 4-OHT-ER per ERE. When DNA footprinting was used to determineER-ligand binding to adjacent EREs, identical high-affinity binding wasobserved for unliganded dimeric ER or ER bound to E2, 4-OHT, andtamoxifen aziridine. These results suggest that ligand-inducedconformation changes primarily affect how the ER interacts with thecomponents of the transcription initiation complex thereby mediatingtranscriptional activation.

The compounds of the present invention were evaluated by comparingbreast cancer cell lines MCF-7 (ER-positive) and MDA-MB435 (ER-negative)with noncancerous breast epithelial cells (MCF-10). Those compounds thatshowed a high level of antiproliferative activity against tested breastcancer cell lines, but not against normal breast epithelial cells wereevaluated for in vitro mechanism of action by looking at their effectsagainst cellular growth factors, Epidermal Growth Factor (EGF), andTransforming Growth Factor (TGF-alpha).

Biological activities (anti cancer) were measured under the followingconditions:

1. A dose response curve for compounds, series I, II, III, IV, over aconcentration range of 1 mM to 1 nM was prepared in order to detect thedoses where possible biological activity might exist. Biologicalactivity was determined by relative LC₅₀ values of these agents. Thosecompounds that exhibited significant decreased cell viability within theestablished concentration range parameters were examined for theirrelative biological activity and potency against known antibreast canceragents tamoxifen, genistein, and doxyrubicin.

2. The compounds in series I, II, III, and IV were examined against twodifferent types of breast cancer cell lines which included estrogenreceptor positive (MCF-7) and estrogen receptor negative (MDA-MB435)cell lines. This examination allowed our laboratory to determine whetherthe novel agents exhibit relative selectivity for estrogen stimulatedbreast cancer cell lines versus estrogen non stimulated breast cancercell lines.

3. Toxicity of the compounds for normal breast epithelial cells wereexamined by comparing LC₅₀ values in normal breast epithelial cell line(MCF-10) against LC₅₀ values in cancer cell lines. LC₅₀ is theconcentration that is lethal to 50 percent of the cells. LC₅₀ valueswere estimated from a dose response graph.

4. The possible mechanism of action of these compounds was explored byexamining the ability of these agents to reverse growth factorstimulated proliferation. The ability of these compounds to attenuateEGF and TGF stimulated growth was tested.

By examining these parameters, our laboratory was able to determinewhether these novel agents possess any antiproliferative activityagainst specific breast cancer cell lines MCF-7 (ER-positive) andMDA-MB435 (ER-negative) or toxicity towards noncancerous breastepithelial (MCF-10). Further, by pursuing this examination ourlaboratory was able to determine to some extent that certain structuralmodifications enhance the biological activity and/or potency of thesecompounds.

Two human breast cancers cell lines (MCF-7 and MDA-MB435) and a normalbreast epithelial cell line (MCF-10) were obtained from ATCC (AmericanType Culture Collection) to test against the series of compounds and thedata were examined for the compounds' relative antiproliferationprofiles. All of these cell lines were derived from epithelial tissue.These cell lines were selected because they allowed our laboratory toinvestigate the effects of the compounds in: 1) breast neoplasms whosegrowth is stimulated by the presence of estrogen, MCF-7 (ER-positive);2) Breast neoplasms whose growth is not stimulated by estrogen MDA-MB435(ER-negative); and 3) A non-cancer cell line (MCF-10) to comparerelative toxicities of the compounds.

It has always been difficult to obtain cultures of breast tumor cellsfrom primary mammary tumors because the presence of fibroblasts, andfatty and lymphocytic tissue make it difficult to isolate the mostlyepithelia breast tumor cells. Even when such cells are isolated, thereis only a low number of viable tumor cells in solid tumors. Cell lineshave been successfully established from pleural effusions, however.Pleural effusions arise from metastasizing tumors and are found in thecavity between lung and the chest. The fact that the tumors aremetastasizing probably indicates the presence of more active cells, andthe cells in the pleural effusion may somehow have adapted to a liquidenvironment.

Most of the human breast cancer cells in continuous culture are ofepithelial origin. MCF-7 cells were obtained by growing a primaryculture from a pleural effusion of a patient with an infiltrating ductalcarcinoma. This primary culture continuously produced free-floatingcells that were used to initiate the stable MCF-7 cell line. This cellline possesses high affinity for estradiol, which stimulates its growth.It also has the insulin receptor but there are conflicting reports aboutgrowth induction by insulin.

General Screening Results

The following sections present the findings of the biological screeningassays for the series I, II, III, and IV compounds. These experimentsdealt with non-stimulated cell growth and the ability of the syntheticcompounds to alter the viability of these cells.

EXAMPLE 1

Series I compounds were chalcone like-compounds where the A ring hasbeen replaced by the adamantyl moiety. Various concentrations of thesecompounds were added to cancer cell lines and biological activity wasmeasured as detailed in the experimental section.

Series I compounds demonstrated limited activity on the viability ofMCF-7 cell, with the exception of 43f which exhibited a pronouncedeffect on cell viability at concentrations greater than 10⁻⁵ M. At theseconcentrations, 43f decreased cell viability 20 percent. These series ofcompounds produced similar effects on MD-MBA435 cells. However, 43faltered cell viability only at much higher concentrations than requiredfor MCF-7 cells.

EXAMPLE 2

Series II compounds are chalcone like-compounds with a modification onthe A ring and the B region, where the A ring has been replaced by theadamantyl moiety and the addition of a double bond to the B region.Various concentrations of these compounds were added to cancer celllines and biological activity was measured as detailed in theexperimental section.

Series II compounds demonstrated little activity in altering theviability of MCF-7 cells. 45a produced a small attenuation in theviability of these cells, while other members of these series were ineffective.

EXAMPLE 3

Series III compounds are chalcone like-compounds where the A ring hasbeen replaced by the adamantyl moiety, and the C ring replaced by aheterocyclic group. A dose response analysis was performed for thesecompounds to determine their cytotoxic properties as specified in theexperimental section.

Compounds, (47a-e), were found to show significant anti-cancer activityagainst the MCF-7 cell lines. The LC₅₀ values for series III (50 μM, 40μM, 50 μM, 5 μM, and 0.5 μM respectively) were consistently lower thanthe LC₅₀ values of known anti-breast cancer agents: Tamoxifen,Doxyrubicin, and Genestein, indicating higher potency. As demonstratedherein, the compounds in series III were consistently more effective atlower concentrations (<10 μM) than the known anti-cancer agents withcompounds 47-c and 47-e showing the greatest efficacy. Three compounds(47a-c) in series III demonstrated significant anti-cancer activityagainst MDA-MB435 cells, with LC₅₀ values of 1 μM, 5 μM, and 50 μMrespectively. Known anti-cancer agents tamoxifen and Genestein weremeasurably less potent in MDA-MB435, with LC₅₀ values of 0.5 mM and 5 mMrespectively. (No measurable LC₅₀ was obtained for Doxorubicin in theconcentration range measured).

These three compounds demonstrated greater activity compared toDoxyrubicin and Genestein at concentrations between 10 nM and 0.1 μM,and greater potency at higher concentrations. Tamoxifen demonstratedsimilar activity to our synthesized compounds only at concentrationsabove 0.1 mm, but was ineffective at lower concentrations. Compounds47-d and 47-e proved to be less promising against MDA-MB435 cells. Forcompound 47-e, an LC₅₀ value of ca 5-mM was obtained, for compound 47-d,no LC₅₀ value was obtained in the concentration range measured.Tamoxifen and Doxyrubicin were measurably less potent against thesecells (LC₅₀ values of 0.5 mM and 5 mM, respectively). In theconcentration range tested, no LC₅₀ was obtained for Genestein.

The relative toxicity of the synthesized compounds (47a-e) againstnormal breast cells was studied by exposing normal breast epithelialcell line MCF-10 to our synthesized compounds in concentration rangesfrom 1 nM to 1 mM. These cells were likewise exposed to equivalentconcentrations of Tamoxifen, Genestein, and Doxyrubicin. Series IIIsynthesized compounds did not significantly decrease the average percentcell viability, which was maintained near one hundred percent, at lowerconcentration of these compounds. At concentrations lower than 10⁻⁷ M,these compounds appeared to enhance the viability of cells.

EXAMPLE 4

Series IV synthesis was directed to chalcone compounds where the A ringhas an adamantyl moiety. A dose response analysis was performed for acompound within this series to determine its cytotoxic properties, asspecified in the experimental section. This compound showed nobiological activity at low concentrations, but exhibited a smalldecrease in cell viability at concentration greater than 10⁻⁵ with MCF-7cells.

Chalcones and isoflavone compounds, such as Genestein, have been citedto exhibit anti-breast cancer activity, mostly against ER positivebreast neoplasms. Antiestrogens, such as tamoxifen, are effective incontrolling the growth of estrogen receptor positive breast tumors.Doxyrubicin, an alkylating agent is a general anti cancer drug that canbe administered for the treatment of breast cancer.

All of these antineoplastic agents were selected as standards to comparewith the novel chalcone-substituted adamantane compounds (47a-e) fortheir relative anticancer activity against human breast cancer celllines MCF-7 (ER-positive) and MDA-MB435 (ER-negative). The compoundswere also examined for their relative toxicity against normal breastepithelial cell line MCF-10. In order to detect the concentrations atwhich the (47a-e) compounds exhibited an anti breast cancer activity, aconcentration response curve was generated from 1 mM to 1 nM. Thisconcentration range was able to give us a broad spectrum of ability todecrease the percent of cell viability of the two human breast cancercell lines.

In the initial evaluation novel adamantane substituted chalconederivatives, five of these compounds exhibited significant anti-breastcancer activity against MCF-7 cells, over the tested concentrationranges. Compounds (47a-e) decreased the % cell viability of MCF-7 by 50%with LC₅₀ values of 5 mM, 1 mM, 0.5 mM, 0.5 mM, and 50 respectively.47-e (50 μM) proved to be the most potent analog against MCF-7 cellscompared to the other active analogs. This suggests an important moietymodification in the chalcone skeleton structure that can enhance thepotency of the chalcones-like derivatives. Genestein is an isoflavonethat is a precursor to chalcone molecules. Therefore the comparison of(47a-e) compounds to Genestein for anti breast cancer activity wasparamount.

The results indicated that the novel (47a-e) compounds were moreeffective than Genestein in increasing the percent cell death of MCF-7cells. This suggests that the adamantane substitutions were an importantmodification to the chalcone skeleton in terms of enhancing biologicalactivity. LC₅₀ concentrations demonstrated that 47-e was more potentthan tamoxifen, inducing 50% cell death in MCF-7 cells at concentrationsof 50 μM and 0.5 mM, respectively. Two compounds, 47-c and 47-d, hadcomparable LC₅₀ values with tamoxifen. At high concentrations (1 mM) alleight compounds, except 47-b and genestein, induced cell death in excessof 70% in MCF-7 cells, indicating potential cytotoxic effects.

Although the LC₅₀ value of 47-e only represented a 10-fold increase in %cell death over that obtained for tamoxifen, it neverthelessdemonstrated a consistent concentration response curve in contrast totamoxifen's sudden change in slope. At concentrations lower than 1 nM,47-e was significantly more potent than tamoxifen. Compounds 47-d and47-c likewise showed a graded response curve, and exceeded tamoxifenpotency below 0.1 mM.

Compound 47e was also found to be more potent than doxorubicien againstMCF-7 cells at LC₅₀ concentrations of 10 μM and 0.5 mM, respectively.47-c and 47-d were equally potent as doxorubicien. Doxorubicien has beenreported to exhibit general anti breast cancer activity, with nospecificity for ER-positive vs. ER-negative breast cancer neoplasms.Therefore, doxorubicin serves as a neutral anti neoplastic agent in thein vitro anti breast cancer screening procedure.

Compounds 47 a-c,e also exhibit anticancer activity against human breastcancer cell line MDA-MB435, with LC₅₀ values of 5 μM, 5 μM, 5 μM, and 1mM, respectively. The anti breast cancer activity exibited by 47 a-cagainst MDA-MB435 cells represented a 1000 fold increase in potency overboth Genestein and Tamoxifen (each with LC₅₀ values of 5 mM). The thirdknown anticancer agent, Doxorubicin demonstrated no detectable LC₅₀value in the concentration range measured.

These results are not surprising, considering that Genestein is found tobe selective for estrogen receptor positive breast cancers, and thattamoxifen has been found to induce sensitivities toward a subpopulationof ER-positive breast tumors. However, based on the enhanced potency ofthese chalcone-like compounds (series III compounds) vs. structurallysimilar Genestein, the substitutions of adamantane moieties indicate animportant substitution in designing potential anti breast cancer agentswith chalcone like skeleton structures.

By LC₅₀ values, 47e demonstrated a significant decrease in percent cellviability against MDA-MB435 cells compared to its efficacy towards MCF-7cells. Also, 47d caused a significant decrease in percent cell viabilityin MDA-MB435 cells as compared to MCF-7 cells, with undetectable LC₅₀values in the tested concentration ranges. These significant changes inspecificity for ER-positive and ER-negative human breast tumors couldrepresent important structural modifications with chalcone skeletalstructures that changes allows this group of chalcones to be directedtowards ER-negative vs. ER-positive breast neoplasms.

Compounds 47a-e were all tested for their relative toxicities against anormal breast epithelial cell line MCF-10. At the highest concentrationstested (1 mM), none of the series III compounds decreased the % cellviability of MCF-10 cells below 80% with average values of 103, 124,102, 94, and 98, respectively. This effect demonstrates the selectiveability of these novel compounds to significantly affect the growth ofcancer cells without interrupting the mechanisms involved withproliferation of normal breast epithelial cells.

In summary, the results indicated that the novel chalcone-like compounds(47a-e) significantly increased (p<0.01) the cell death of both humanbreast cancer cell lines MCF-7 (ER-positive), and MDA-MB435(ER-negative), without altering the viability of normal breastepithelial cell line MCF-10.

Antiestrogens and Growth Factors

During the past 20 years, considerable focus has been put on themechanisms whereby cells modulate the growth stimulus or stop growingwhen the task of replication is complete. The identification of familiesof stimulatory or inhibitory growth factors that affect the same cell(autocrine factors) or adjacent cells (paracrine factors) hasrevolutionized the concepts of hormonal regulation. The ideas have beentranslated during the past decade from general physiology to be appliedto cancer control.

Transforming Growth Factor α

Estrogen is believed to increase the production of TGFα and, throughautocrine activation of the epidermal growth factor receptor, encouragereplication. However, TGFα alone cannot substitute for estrogen. MCF-7cell transfected with the cDNA for TGFα are not tumorigenic in athymicmice.

Studies by Wakeling and colleagues compared the ability the antiestrogentamoxifen or its active metabolite, 4-OHT, to attenuate the stimulatoryeffects of TGFα on MCF-7 cells. They showed that when MCF-7 cells aretreated with TGFα, tomaxifen partially blocks the stimulatory effect inthe absence of E2. In contrast, studies using EGF instead of TGFα showedthat antiestrogens could not block the actions of the growth factor, andalso that antiestrogens could not block the paracrine influence ofER-negative cells from stimulating MCF-7 cells in vitro. In addition, itis known that estrogens can induce the expression of TGFα inestrogen-responsive breast cancer cell lines, whereas antiestrogensgenerally decrease TGFα expression in vitro and in vivo.

TGFα apparently is essential for estrogen-stimulated,anchorage-independent growth. TGFα or epidermal growth factor receptorantibodies can negate the E2-stimulated, anchorage-independent growth ofMCF-7 cells on soft agar. The antibodies did not affect progesterone orprolactin.

The effects of antiestrogens on TGFα expression in vivo have not beenstudied extensively; however, one study shows that tamoxifen is capableof down-regulating tumor TGFα expression in postmenopausal women withER-and PR-positive disease but not in women with ER-and PR-negativedisease.

The regulation of TGFα remains unclear. A few putative half-site EREshave been identified in the promotor region of the TGFα gene, but othersites in the promotor region are required for gene activation.Constructs of the EREs alone do not appear to respond to estrogen actionunless the cells are super-transfected with ER. By contrast, ER-negativecells that are stably transfected with ER will induce TGFα mRNA inresponse to estrogen.

Perhaps, most interesting is the effect of antiestrogens. Raloxifeneacquires the ability to initiate TGFα synthesis when ER-negative cellsare stably transfected with a 351-mutant ER. However, in ER-negativetransfectants containing wild-type ER, raloxifene is a completeantiestrogen. 4-OHT acts as an estrogen (induction of TGFα) in bothwild-type and mutant ER stable transfectants. Because antiestrogensproduce different effects in transfectants expressing wild-type ormutant ER, and because 4-OHT and estrogen can both initiate TGFα mRNAtranscription equally, this provides a unique model to determine whichproteins associate with the antiestrogen-ERE complex to make it sopromiscuous.

Transforming Growth Factor β

The TGFβ family of inhibitory polypeptides consists of three or more 25kDa members, which are able to homo-or heterodimerize to form complexesthat interact with the TGFβ receptor (TGFβR). These peptides areimplicated in breast cancer and have been found to be over-expressed andcorrelate with tumor progression. TGFβ binds to any of the differentcharacterized TGFβRs. The receptor consists of a heterodimeric complex,one part of which is a binding protein that is unable to signal andanother part that is believed to transduce signals to the cell throughserine-threonin kinase activity. The ability of TGFβ to promote tumorprogression is counterintuitive because TGFβ usually produces eithergrowth inhibition or differentiation, neither of which are involved intumor progression. Further study clearly is needed in vivo to determinewhat cooperating factors dictate the effects of TGFβ on different celltypes, because the results may be critical to understanding the successor failure of antiestrogen therapy.

The effect of tamoxifen on the production of TGFβ is an area of greatinterest. Elucidation of a mechanism could provide an explanation forthe cell cycle effects of tamoxifen in ER-positive cells and alsoprovide an explanation for the sporadic reports of the success oftamoxifen treatment in ER-negative breast cancer. Much work has beencompleted in cell culture but there are important translational aspectsof the research that are relevant in understanding the action oftamoxifen.

Tamoxifen has a direct effect on the production of TGFβ in breast cancercells. TGFβ expression increases in MCF-7 cells, and further study hasshown a differential activation of members of the TGFβ family. However,the results are variable. Some studies report an increase in TGFβ-2 withtamoxifen, whereas others demonstrate rises in TGFβ-1. This observationhas been translated to the clinic. Patients that respond to tamoxifentherapy show increases in TGFβ-2 plasma levels. Knabbe's study suggeststhat the results of measuring either TGFβ-1 levels (whichtranscriptionally activates TGFβ-2) or TGFβ-2 in the plasma can be usedas a predictive test for the efficacy of tamoxifen therapy.

Some support for the central role of TGFβ-2 comes from sampling tumorsdirectly. When TGFβ mRNA levels from ER-positive breast tumors weremonitored before and during tamoxifen therapy, the results werevariable. Changes in TGFβ-1 and TGFβ-2 did not correlate with tamoxifentreatment, but there was a significant correlation between treatment andchanges in TGFβ-2 in some tumors. The authors concluded that response totamoxifen therapy might be mediated through an increase in theexpression of a particular TGFβ iso-form.

The effect of tamoxifen on ER-negative tumors is far more controversial.Perry and coworkers have compared and contrasted the effect of tamoxifenon the induction of TGFβ-1 in an ER-positive and an ER-negative cellline. After long-term treatment, the expression of TGFβ-1 increased,independent of ER status, but an accumulation of cells in G1/G0 and anincrease in apoptosis occurred concurrently. This conclusion tends tosupport a model of the direct effect of tamoxifen on ER-negative cells.

By contrast, it is possible that the growth of an ER-negative cell iscontrolled by a paracrine mechanism. Perhaps the ER-positive cellproduces TGFβ in response to tamoxifen, but the secreted growth factorstops the growth of the adjacent ER-negative cells. It is known thatER-negative breast cancer cells have a high density of TGFβ receptors,and the cells respond to TGFβ by growth inhibition. The hypothesis thatan ER-positive cell can control the growth of ER-negative cells duringtamoxifen therapy has been demonstrated in vitro. However, this has notbeen possible to test in animal models. Different mixes of ER-positiveand ER-negative cells were inoculated into athymic animals and treatedwith the antiestrogen toremifene. Regrettably, in model, theantiestrogen was unable to control heterogeneous tumor growth.

However, the laboratory finding that tamoxifen can induce TGFβ infibroblasts has introduced a new mechanistic dimension to understand thecontrol of ER-negative disease by tamoxifen. Clearly, if TGFβ can beinduced in the supporting stromal cells of a breast cancer tumor duringtamoxifen therapy, the paracrine growth inhibitor could control theproliferation of ER-negative cells. Butta and coworkers found that TGFβproduction increases in stromal cells during tamoxifen therapy. Althoughthese data illustrate that a complex cellular conversation occurs toregulate cell growth, the fact that tamoxifen is not usually successfulin ER-negative disease means that the pathways are not necessarilydominant. Nevertheless, the realization that TGFβ can act both as agrowth inhibitor and as a growth stimulator may ultimately make thepathways important to explain tamoxifen failure.

In summary, the past decade has seen an elucidation of the role of bothpositive and negative growth factors in estrogen-stimulated growth.Although each effect of tamoxifen on the growth factor system may initself be small, it is possible that the combined actions of tamoxifenare responsible for the benefits documented with tamoxifen in clinicalpractice.

Screening Using the Growth Factor Protocol

The mechanism of the specificity for breast cancer cells vs. normalbreast epithelial cells has not been established. In order to elucidatethe mechanisms, by which these novel series III compounds were able toexert their effects against both MCF-7 and MDA-MB435 cells, the twocells were examined for common receptors that might be involved in cellproliferation. It is known that both cell lines expressed epidermalgrowth factor receptors (EGFR). Also both cell lines are sensitive toepidermal growth factor (EGF) ligands and Transforming Growth Factoralpha (TGF-α).

There have been several studies that have examined the relative changesin expression of EGF, EGFR, and TGF-α in estrogen receptor positive vs.estrogen receptor negative breast cancer cell lines. These changes ingrowth factor expression were correlated with the stimulation orinhibition of cellular proliferation. Also there have been severalstudies that demonstrate that the isoflavone class of compounds exerttheir anti breast cancer effects by altering the EGF, and EGFR proteins.Because of the structural similarity of these isoflavones and chalconesto naturally occurring estrogens, it was initially suggested that theseisoflavones might prevent hormone-dependent breast and prostate cancersby virtue of their potential estrogen-antagonist activity. Human breastcancer (MCF-7) cells, when placed ectopically in ovariectomized adultrats, grew at a faster rate in rats fed genestien-containing diets thanin those fed control diets, yet another indication of the estrogenicactivity of genistein.

In cell culture, genistein inhibited proliferation of human breast andprostate cancer cells stimulated by epidermal growth factor (EGF)independently of whether the cells expressed estrogen receptors. In thepresence of 17β-estradiol, genistein induced no additional proliferationat any concentration examined. Instead, at concentration >5 μmol/L itcaused a dose-dependent reduction in the 17β-estradiol-stimulated cellproliferation. These data indicated that genistein may inhibit cellproliferation by mechanisms other than classicalestrogen-receptor-mediated pathway. Genistein cannot, therefore, beviewed simply as either an agonist or antagonist of estrogen.Understanding the biological effects of genistein in vivo also requiresan appreciation of all of its mechanisms of action. Therefore, the novelseries III compounds were evaluated for their relative abilities toreverse the growth stimulatory effects of EGF and TGF.

Concentration of genistein that inhibit proliferation of human breastcell lines do not effect phosphorylation of EGFR or other targetproteins which are normally stimulated by EGF. Normal human mammaryepithelial (HME) cells (which do not express estrogen receptor)stimulated by EGF are exquisitely sensitive to growth inhibition bygenistein without any inhibition of EGF-R tyrosine autophosphorylation;the concentration at which cell growth is inhibited by 50% (IC50) is 1μmol/L. These data strongly suggest that genistein affects these cells(and therefore other systems) by mechanisms other than inhibition of PTKactivity.

In a glioblastoma model in vitro, both invasiveness and proliferation ofglioblastoma cells were dependent on EGF, but the invasiveness wasblocked by genistein at concentrations that did not affect proliferationor EGF-R autophosphorylation. These data provide compelling evidence of2 subsets of activities modulated by EGF-R, only one of which isaffected by genistein.

Both MCF-7 and MDA-MB435 cells contain EGF-R and TGF-R. In an effort toexamine the role of EGF-R and TGF-R in the proliferation of MCF-7 andMDA-MB435, these cells were stimulated to growth stimulatory at 50percent (GS₅₀) values. The cells were treated with the synthesizedcompound 47a at concentrations ranges of 10⁻⁹ to 10⁻³ M.

EGF-Serum and Non-Serum Stimulated Growth of MCF-7 Cells

The results indicated that EGF-serum stimulated growth of MCF-7 cellswas significantly (p<0.02) reversed back to control values by 47a at 10μM. EGF-non serum stimulated growth was also significantly (p<0.102)reversed back to control values by 47a at IC₅₀ values of 50 μM.

TGF-α Serum and Non-Serum Stimulated Growth of MCF-7 Cells

The next experiment was performed to examine the possible role of TGF-αin the proliferation of MCF-7 cells. The results indicated that neitherTGF-α serum stimulated nor non-serum stimulated growth of MCF-7 cellssignificantly reversed by 47a at concentration ranges of 10⁻⁹ to 10⁻³ M.

In evaluating the ability of 47a to inhibit the EGF growth stimulationof MCF-7 cells, compound 47a significantly reversed the growthstimulatory effects of EGF, but not TGF-α.

This could possibly indicate a selective mechanism by which this drugexerts its inhibitory effects against MCF-7 cells. The results indicatethat the EGF serum and non-serum stimulated growth of MCF-7 cells wereinhibited by 47a in a similar manner. This could indicate that theinhibition seen by EGF-serum stimulated growth was mainly due toinhibition of EGF-stimulation. The fact that there was little or noreversal on TGF-α serum or non-serum growth stimulation indicates theselectivity of 47a for EGF in serum stimulated growth.

TGF-α and EGF Serum and Non-Serum Growth Stimulation of MDA-MB 435Cells.

Compound 47a was evaluated for its relative ability to reverse the GS₅₀stimulation of MDA-MB 435 cells by TGF (1 ng/ml). The results indicatedthat the TGF significantly (p<0.02) reversed the serum and non-serumstimulated growth of MDA-MB 435 cells back to control values at 50 μMand 10 μM, respectively.

Neither EGF (1 ng/ml) serum nor non-serum stimulated growth of MDA-MB435cells was reversed by to control values at concentration ranges of 10⁻⁹to 10⁻³ M. This data seems to indicate the relative importance of theTGF-R in MDA-MB435 cells.

Comparing the TGF-α serum to non-serum profiles seems to imply that 47ais able to decrease the cell viability of MDA-MB435 cells by interferingwith the TGF-α mediated growth stimulation of MDA-MB435 cells.

The data seems to suggest that one of the possible mechanisms by which47a might decrease the cell viabilities of the two human breast cancercell lines is to reverse the epithelial growth stimulatory effectsinduced by growth factors EGF and TGF-α. Literature suggest that thesefactors play a key role in the proliferation of epithelial cells. Atconcentration ranges of 1.00 to 50 μM, the compound 47a reversal effectsback to control values correlate with LC₅₀ values obtained by drugtreatment. Although this reversal may not indicate the total mechanismby which the series III synthesized compounds exert their effects on thehuman breast cancer cell lines, but they approximate a quantitativepossibility for their relative mechanism of action.

The results of testing the compounds of the present invention indicatethat alterations in EGF-R stimulation is a key mechanism by which seriesIII compounds decrease the cell viability of MCF-7 cells. This is inagreement with current literature, which indicates an importantconnection between estrogen and epidermal growth factors.

In an effort to elucidate possible mechanisms of action that oursynthesized compounds exhibited against MCF-7 and MDA-MB435 breastcancer cell lines, 47a (the parent compound) was evaluated for itsrelative ability to reverse the growth stimulation at 50% of MCF-7 andMDA-MB435 cells by serum and non-serum stimulated epidermal growthfactor (EGF), and by serum and non-serum stimulated transforming growthfactor (TGF-α). In all cases 10 ml of growth factor was administered ata concentration of 1 ng/ml. Compound 47a was found to be consistentlyeffective in reversing the growth of both cell lines. The reversalvalues are summarized in Table 6.

TABLE 6 LC₅₀ Data for Administered Drug Growth Serum Non-Serum Cell LineFactor Stimulated Stimulated MCF-7 EGF 1 μM 5 μM TGF 5 μM 50 μMMDA-MB435 EGF 5 Mm 7 mM TGF 1 μM 2 μM

Based on these preliminary results, the data indicated that a group ofnovel chalcone-like compounds were synthesized that exhibited a broadrange of anti-cancer activity with significant reductions in cellviabilities against both human breast cancer cell lines MCF-7 andMDA-MB435. Only compounds (47a-e) were found to be active against theabove cell lines. These agents were found to be as potent, and in somecases, more potent than known anti-breast cancer agents. These compoundsexhibited the ability to reverse the stimulatory effects of knowntyrosine kinases EGF and TGFα. This suggests that (47a-e) compoundsmight be exerting their anti-cancer effects against MCF-7 and MDA-MB435cells by blocking the growth stimulatory effects of EGF and/or TGFα.These results indicate the importance of structural modifications ofchemically similar drugs towards improving the efficiency and potency ofpotential antineoplastic agents.

Genistein's effects on cells, including inhibition of proliferation,induction of differentiation, apoptosis, and arrest of cells at cellcycle checkpoints are reminiscent of those of TGFβ1. This peptide growthfactor was identified as a major factor that regulates eukaryotic cellproliferation by attenuating passage through cell cycle check-points.Exposure to genistein caused either synthesis and secretion of TGFβ1 bythe HME cells or secretion of the preexisting intracellular pool ofTGFβ1. The growth inhibitory effects of tamoxifen were nearly identicalto those of genistein involving TGFβ1 on the growth of human breastcancer MCF-7 cells. For both compounds, the inhibition of cellproliferation was correlated with increased amounts of TGFβ1 in theculture medium and was blocked by antibodies against TGFβ1 addedexogenously to the culture medium.

Humans, and other animals, in particular, mammals, suffering from breastcancer or other proliferative disorders can be treated by administeringto the patient an effective amount of one or more of theabove-identified compounds or a pharmaceutically acceptable salt orderivative or salt thereof in a pharmaceutically acceptable carrier ordiluent. The active materials can be administered by any appropriateroute, for example, orally, parenterally, intravenously, intradermally,or subcutaneously.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount without causing serious toxic effectsin the patient treated. A preferred dose of the active compound for allof the above-mentioned conditions is in the range from about 0.5 to 500mg/kg, preferably 1 to 100 mg/kg per day. The effective dosage range ofthe pharmaceutically acceptable salt or derivatives can be calculatedbased on the weight of the parent compound to be delivered. If thederivative exhibits activity in itself, the effective dosage can beestimated as above using the weight of the derivative, or by other meansknown to those skilled in the art.

The compound is conveniently administered in any suitable unit dosageform, including but not limited to one containing 1 to 3000 mg,preferably 5 to 500 mg of active ingredient per unit dosage form. Anoral dosage of 25-250 mg is usually convenient.

The active ingredient should be administered to achieve peak plasmaconcentrations of the active compound of about 0.1 to 100 μM, preferablyabout 1-10 μM. This may be achieved, for example, by the intravenousinjection of a solution or formulation of the active ingredient,optionally in saline, or an aqueous medium or administered as a bolus ofthe active ingredient.

The concentration of active compound in the drug composition will dependon absorption, distribution, inactivation, and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed invention. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

Oral compositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The active compound or pharmaceutically acceptable salt or derivativethereof can be administered as a component of an elixir, suspension,syrup, wafer, chewing gum or the like. A syrup may contain, in additionto the active compounds, sucrose as a sweetening agent and certainpreservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable salt or derivativesor salts thereof can also be administered with other active materialsthat do not impair the desired action, or with materials that supplementthe desired action, such as other antiproliferative agents.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Combination Therapy

Compounds of the present invention may be administered in combinationwith other anti-proliferative agents. When a combination of two or moreanti-proliferative or potentially anti-proliferative agents is assayed,the results may indicate less inhibition of proliferation than whatwould be expected if the effects of the individual agents were additive,or the effects may represent the mathematical product of the expectedeffects of the two agents (additive inhibition). Alternatively, theinhibition actually observed experimentally may be greater than whatwould be expected as a simple product of the effects of the two agents.Such synergistic anti-tumor or antiproliferative effect is highlydesirable. This synergistic effect of the compounds with otherchemotherapeutic agents in the treatment of tumors, and especially ofbreast cancer, is contemplated by the present invention.

The chemotherapeutic agents used in combination with the compounds ofthe present invention include tamoxifen, toremifene, idoxifene,droloxifene, TAT-59, LY117018, raloxifene, genistein, doxyrubicin,Taxol, Taxotere, aredia, arimidex, navilbine, busulfan, cisplatin,cyclophosphamide (Cytoxan), dacarbazine, ifosfamide, interferon,mechlorethamine (Mustargen), melphalan, carmustine, lomustine,5-fluorouracil, methotrexate, gemcitabine, cytarabine (Ara-C),fludarabine, bleomycin, dactinomycin, daunorubicin, idarubicin,paclitaxel, docetaxel, etoposide, vinblastine, vincristine, vinorelbine,prednisone, dexamethasone, and herceptin.

Whereas this invention has been described in detail with particularreference to its most preferred embodiments, it is understood thatvariations and modifications can be effected within the spirit and scopeof the invention, as described herein before and as defined in theappended claims. The corresponding structures, materials, acts, andequivalents of all means plus function elements, if any, in the claimsbelow are intended to include any structure, material, or acts forperforming the functions in combination with other claimed elements asspecifically claimed.

1. A compound of the formula:

wherein R₁ is Ad- or Ad-(L1)n-, wherein n is 0 or 1, Ad is adamantyl,and L1 is a linking group selected from the group consisting of C1-6alkylene, C1-6 cycloalkylene, and C1-6 arylene; Y is H; and R₂ isCY1═CHR3, wherein Y1 is H, C1-6 alkyl, aryl, or halo; and R3 is aryl,aryl optionally substituted by X, or HET; X is hydrogen, straight chainor branched C1-6 alkyl, halo, amino, C1-6 alkyl amino, C1-6 dialkylamino, pyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, C1-6 alkoxy,C1-6 aralkoxyl, aryl, C1-6 aralkyl, nitro, cyano or a phosphoruscontaining group; and HET is optionally substituted pyrrolidinyl,piperadinyl, morpholinyl, piperazinyl, pyrrolyl, pyridinyl, andpyridazinyl, quinolinyl, or thiophenyl, and wherein the substitutuent isX; or a pharmaceutically acceptable salt or derivative thereof.
 2. Thecompound according to claim 1, having the formula:

wherein R₁ is Ad- or Ad-(L1)n-, wherein n is 0 or 1, Ad is adamantyl,and L1 is a linking group selected from the group consisting of C1-6alkylene, C1-6 cycloalkylene, and C1-6 arylene; Y is H; R₂ is CY1═CHR3,wherein Y1 is H, C1-6 alkyl, aryl, or halo; R3 is aryl, or aryloptionally substituted by X; and X is hydrogen, straight chain orbranched C1-6 alkyl, halo, amino, C1-6 alkyl amino, C1-6 dialkyl amino,pyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, C1-6 alkoxy, C1-6aralkoxyl, aryl, C1-6 aralkyl, nitro, cyano or a phosphorus containinggroup; or a pharmaceutically acceptable salt or derivative thereof. 3.The compound according to claim 1 having the formula:

wherein Y is H, C1-6 alkyl, aryl, or halo; and X is hydrogen, straightchain or branched C1-6 alkyl, halo, amino, C1-6 alkyl amino, C1-6dialkyl amino, pyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, C1-6alkoxy, C1-6 aralkoxyl, aryl, C1-6 aralkyl, nitro, cyano or a phosphoruscontaining group; or a pharmaceutically acceptable salt or derivativethereof.
 4. The compound according to claim 1 having the formula:

wherein X is H or OCH₃, and Y is H, CH₃ or Cl.
 5. A pharmaceuticalcomposition comprising an anti-proliferative effective amount of acompound of claim 1 having the formula:

wherein R₁ is Ad- or Ad-(L1)n-, wherein n is 0 or 1, Ad is adamantyl,and L1 is a linking group selected from the group consisting of C1-6alkylene, C1-6 cycloalkylene, and C1-6 arylene; Y is H; R₂ is CY1=CHR3,wherein Y1 is H, C1-6 alkyl, aryl, or halo; R3 is aryl, or aryloptionally substituted by X; and X is hydrogen, straight chain orbranched C1-6 alkyl, halo, amino, C1-6 alkyl amino, C1-6 dialkyl amino,pyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, C1-6 alkoxy, C1-6aralkoxyl, aryl, C1-6 aralkyl, nitro, cyano or a phosphorus containinggroup; or a pharmaceutically acceptable salt or derivative thereof, incombination with a pharmaceutically acceptable carrier.
 6. Apharmaceutical composition comprising an anti-proliferative effectiveamount of a compound of claim 1 having the formula:

wherein Y is H, C1-6 alkyl, aryl, or halo and X is hydrogen, straightchain or branched C1-6 alkyl, halo, amino, C1-6 alkyl amino, C1-6dialkyl amino, pyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, C1-6alkoxy, C1-6 aralkoxyl, aryl, C1-6 aralkyl, nitro, cyano or a phosphoruscontaining group; or a pharmaceutically acceptable salt or derivativethereof, in combination with a pharmaceutically acceptable carrier.
 7. Amethod for the treatment of a proliferative disorder selected from thegroup consisting of prostate cancer, lung cancer, pancreatic cancer,breast cancer, colon cancer, ovarian cancer, and bladder cancer,comprising administering to a host in need of such treatment ananti-proliferative effective amount of a compound according to claim 1,optionally in combination with a pharmaceutically acceptable carrier. 8.A method for the treatment of a proliferative disorder selected from thegroup consisting of prostate cancer, lung cancer, pancreatic cancer,breast cancer, colon cancer, ovarian cancer, and bladder cancer,comprising administering to a host in need of such treatment ananti-proliferative effective amount of a compound according to claim 1having the formula:

wherein R₁ is Ad- or Ad-(L1)n-, wherein n is 0 or 1, Ad is adamantyl,and L1 is a linking group selected from the group consisting of C1-6alkylene, C1-6 cycloalkylene, and C1-6 arylene; Y is H; R₂ is CY1═CHR3,wherein Y1 is H, C1-6 alkyl, aryl, or halo; R3 is aryl, or aryloptionally substituted by X; and X is hydrogen, straight chain orbranched C1-6 alkyl, halo, amino, C1-6 alkyl amino, C1-6 dialkyl amino,pyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, C1-6 alkoxy, C1-6aralkoxyl, aryl, C1-6 aralkyl, nitro, cyano or a phosphorus containinggroup; or a pharmaceutically acceptable salt or derivative thereofoptionally in combination with a pharmaceutically acceptable carrier. 9.A method for the treatment of a proliferative disorder selected from thegroup consisting of prostate cancer, lung cancer, pancreatic cancer,breast cancer, colon cancer, ovarian cancer, and bladder cancer,comprising administering to a host in need of such treatment ananti-proliferative effective amount of a compound according to claim 1having the formula:

wherein Y is H, C1-6 alkyl, aryl, or halo; and X is hydrogen, straightchain or branched C1-6 alkyl, halo, amino, C1-6 alkyl amino, C1-6dialkyl amino, pyrrolidinyl, piperadinyl, morpholinyl, piperazinyl, C1-6alkoxy, C1-6 aralkoxyl, aryl, C1-6 aralkyl, nitro, cyano or a phosphoruscontaining group; or a pharmaceutically acceptable salt or derivativethereof optionally in combination with a pharmaceutically acceptablecarrier.
 10. A method for the treatment of a proliferative disorderselected from the group consisting of prostate cancer, lung cancer,pancreatic cancer, breast cancer, colon cancer, ovarian cancer, andbladder cancer, comprising administering to a host in need of suchtreatment an anti-proliferative effective amount of a compound accordingto claim 1 having the formula:

wherein X is H or OCH₃, and Y is H, CH₃ or Cl; or a pharmaceuticallyacceptable salt or derivative thereof optionally in combination with apharmaceutically acceptable carrier.
 11. A method for the treatment ofbreast cancer comprising administering to a host in need of suchtreatment an anti-proliferative effective amount of a compound accordingto claim 1 having the formula:

wherein R₁ is Ad- or Ad-(L1)n-, wherein n is 0 or 1, Ad is adamantyl,and L1 is a linking group selected from the group consisting of C1-6alkylene, C1-6 cycloalkylene, and C1-6 arylene; Y is H; and R₂ isCH═CHR3, wherein R3 is aryl, aryl optionally substituted by X, and X ishydrogen, straight chain or branched C1-6 alkyl, halo, amino, C1-6 alkylamino, C1-6 dialkyl amino, pyrrolidinyl, piperadinyl, morpholinyl,piperazinyl, C1-6 alkoxy, C1-6 aralkoxyl, aryl, C1-6 aralkyl, nitro,cyano or a phosphorus containing group; or a pharmaceutically acceptablesalt or derivative thereof in combination with at least one otherchemotherapeutic agent selected from the group consisting of tamoxifen,toremifene, idoxifene, droloxifene, TAT-59, LY117018, raloxifene,genistein, doxyrubicin, Taxol, Taxotere, aredia, arimidex, navilbine,busulfan, cisplatin, cyclophosphamide (Cytoxan), dacarbazine,ifosfamide, mechlorethamine (Mustargen), melphalan, carmustine,lomustine, 5-fluorouracil, methotrexate, gemcitabine, cytarabine(Ara-C), fludarabine, bleomycin, dactinomycin, daunorubicin, idarubicin,paclitaxel, docetaxel, etoposide, vinblastine, vincristine, vinorelbine,prednisone, dexamethasone, and herceptin, optionally in combination witha pharmaceutically acceptable carrier.
 12. A method for the treatment ofbreast cancer comprising administering to a host in need of suchtreatment an effective amount of a compound according to claim 1 havingthe formula:

wherein R₂ is CH═CHR3, wherein R3 is aryl, aryl optionally substitutedby X, wherein X is hydrogen, straight chain or branched C1-6 alkyl,halo, amino, C1-6 alkyl amino, C1-6 dialkyl amino, pyrrolidinyl,piperadinyl, morpholinyl, piperazinyl, C1-6 alkoxy, C1-6 aralkoxyl,aryl, C1-6 aralkyl, nitro, cyano or a phosphorus containing group; or apharmaceutically acceptable salt or derivative thereof in combinationwith at least one other chemotherapeutic agent selected from the groupconsisting of tamoxifen, toremifene, idoxifene, droloxifene, TAT-59,LY117018, raloxifene, genistein, doxyrubicin, Taxol, Taxotere, aredia,arimidex, navilbine, busulfan, cisplatin, cyclophosphamide (Cytoxan),dacarbazine, ifosfamide, mechlorethamine (Mustargen), melphalan,carmustine, lomustine, 5-fluorouracil, methotrexate, gemcitabine,cytarabine (Ara-C), fludarabine, bleomycin, dactinomycin, daunorubicin,idarubicin, paclitaxel, docetaxel, etoposide, vinblastine, vincristine,vinorelbine, prednisone, dexamethasone, and herceptin, optionally incombination with a pharmaceutically acceptable carrier.
 13. A method forthe treatment of breast cancer comprising administering to a host inneed of such treatment an effective amount of a compound according toclaim 1 having the formula:

wherein X is H or OCH₃, and Y is H, CH₃ or Cl; or a pharmaceuticallyacceptable salt or derivative thereof in combination with at least oneother chemotherapeutic agent selected from the group consisting oftamoxifen, toremifene, idoxifene, droloxifene, TAT-59, LY117018,raloxifene, genistein, doxyrubicin, Taxol, Taxotere, aredia, arimidex,navilbine, busulfan, cisplatin, cyclophosphamide (Cytoxan), dacarbazine,ifosfamide, mechlorethamine (Mustargen), melphalan, carmustine,lomustine, 5-fluorouracil, methotrexate, gemcitabine, cytarabine(Ara-C), fludarabine, bleomycin, dactinomycin, daunorubicin, idarubicin,paclitaxel, docetaxel, etoposide, vinblastine, vincristine, vinorelbine,prednisone, dexamethasone, and herceptin, optionally in combination witha pharmaceutically acceptable carrier.